Throughout this application, various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citation for these references may be found at the end of this application, preceding the claims.
This invention comprises a series of new therapeutics and targets for therapeutic intervention in HIV-1 infection. Monoclonal antibodies have been identified that inhibit HIV-1 envelope-mediated membrane fusion and thereby inhibit virus infection. The antibodies were discovered by screening panels of monoclonal antibodies generated by immunizing mice with human cells. The screening was initially performed using a resonance energy transfer (RET) assay of HIV-1 envelope-mediated membrane fusion. Antibodies which inhibited in this assay were further screened for inhibitory activity in a HIV-1 infection assay.
These inhibitory antibodies act by binding to molecules on the surface of cells, which are required for HIV-1 to fuse with and infect target cells. The molecules are either previously unidentified or their role in HIV-1 entry was previously unrecognized. The cell surface molecules are known as accessory molecules, since they are required for virus entry in addition to the HIV-1 receptor, CD4. While CD4 is required for HIV-1 attachment, the accessory molecules are required for the membrane fusion step of entry. These accessory molecules are generally expressed only on human cells, so HIV-1 does not infect non-human cells that have been engineered to express human CD4 (1,3,8,9). Moreover, several groups have shown that it is possible to complement these non-human CD4+ cells by fusing them (using polyethylene glycol) with CD4− human cells, resulting in a heterokaryon which is a competent target for HIV-1 envelope-mediated membrane fusion (2,5).
As discussed above, it is generally accepted that accessory molecules are required for HIV-1 fusion. However, the precise nature of these co-receptors or accessory molecules has not yet been discerned. While some cell surface molecules have previously been implicated as fusion accessory molecules (7,10,11), their role has not been confirmed (4).
In some cases, the fusion accessory molecules are found on a subset of human CD4+ cells and are required for infection by HIV-1 isolates with particular tropisms. For example, macrophage-tropic strains of HIV-1 such as HIV-1JR-FL may have different requirements for accessory molecules compared with T lymphotropic strains such as HIV-1LAI. This phenomenon is, in part, responsible for differences in tropism between HIV-1 strains.
The current invention includes the monoclonal antibodies and the hybridomas which secret them; also their humanized equivalents, single chain antibodies or antigen binding fragments of the antibodies. These antibodies, single chain antibodies or antibody fragments have value as immunotherapeutics or immunoprophylactics for HIV-1 infection. The invention also includes the genes encoding these antibodies, single chain antibodies and antibody fragments. Moreover, the invention includes the accessory molecules recognized by these monoclonal antibodies, or components of these accessory molecules and region(s) of HIV-1 gp120/gp41 that interact with these accessory molecule(s). In addition the invention includes the genes encoding the accessory molecules. The accessory molecules or their fragments have value as therapeutic or prophylactic agents to inhibit HIV-1 infection. They are also valuable as a basis for rationale drug design to identify inhibitors of HIV-1 infection. This invention provides transgenic animals comprising DNA encoding these accessory molecules or fragments thereof. These transgenic animals are useful as animal models of HIV-1 infection. The invention also includes the use of the antibodies and/or accessory molecules in drug screening assays to identify inhibitors of HIV-1 fusion. Finally, the invention includes the inhibitors identified using these drug screens.